Ryanodine receptor (RyR) Ca2+ channel function plays a critical role in Ca2+ homeostasis of striated muscles. Dysfunction of RyRs often result in dysregulation of myoplasmic Ca2+ cycling that has been associated to several myopathies and various forms of arrhythmogenic cardiac disorders. It is currently accepted that ion Ca2+ is the single most important activator of RyRs and modulate channel function through two independent binding sites, one activatory and one inhibitory. However, despite numerous studies, to this date, the location and molecular properties of either Ca2+-binding domain remains largely unknown. This represents a major gap since RyRs have become an important therapeutic target. This study addresses this gap by proposing a comprehensive structural/functional characterization of a newly found Ca2+-binding/regulatory domain of RyRs. The proposal challenges the classic concept of two Ca2+-binding sites by proposing the hypothesis that Ca2+-mediated regulation of RyRs involves the contribution of a new class of Ca2+-binding domain that modulate the Ca2+-activation site and overall Ca2+-cycling properties of the cell. This hypothesis is supported by our recent findings using an innovative RyR3/RyR1 chimeric receptor approach that identified a new discrete functional determinant of RyRs (named as the CBD region) that plays a central role in channel function and Ca2+-cycling regulation of skeletal myotubes. These studies indicate that within the CBD region resides a new class of Ca2+-binding site that is highly conserved among all isoforms of RyRs. The objective of this proposal is to molecularly define and functional characterize this new Ca2+-binding domain and define its role in Ca2+ regulation of adult muscle under normal and myopathic conditions. In Aim-1 we propose a comprehensive structural, biochemical and functional characterization of the new Ca2+- binding domain. Using Fluorescence Spectroscopy, Circular Dichroism and Nuclear Magnetic Resonance in combination with a targeted mutational approach we will map and fully define the new Ca2+-binding motif of RyR1. As functional correlate we will explore the effects of disruption of this Ca2+-binding site on 1 ) Ca2+-sensing properties of full length RyRs using 3H-ryanodine binding and single channels studies and 2) Ca2+-cycling properties of cultured myotubes. In Aim-2 we will explore the role of the new Ca2+-binding site in Ca2+-cycling regulation of adult skeletal muscles using mouse FDB fibers. We will also extend these studies to a myopathic mouse model to explore the translational value of targeted modulation of the new Ca2+-regulatory domain as potential therapeutic vehicle to abate the effects of Ca2+-cycling dysregulation linked to RyR dysfunction. This line of research seeks to generate the molecular basis for future development of new therapeutic approaches against a wide range of skeletal and cardiac myopathies linked to dysregulation of RyRs. Therefore, this application directly relates to the goals of the Division of Musculoskeletal Diseases.